Common anticoagulant therapies primarily aim at inhibiting the procoagulatory factors thrombin (factor IIa, FIIa) and factor Xa (F Xa). A distinction is made between oral anticoagulation with vitamin K antagonists such as Coumadin, for example, which effects inhibition of coagulation factor synthesis, and anticoagulation due to inhibition of the active coagulation factors in the bloodstream. Among the anticoagulants which inhibit or inactivate active coagulation factors in the bloodstream, anticoagulants with direct action and anticoagulants with indirect action are distinguished. Anticoagulants with direct action such as, for example, rivaroxaban, dabigatran or melagatran bind to thrombin or factor Xa and are therefore highly specific. Anticoagulants with indirect action such as, for example, heparins bind to endogenous coagulation factor inhibitors such as antithrombin, for example, and amplify many times their anticoagulatory action.
All anticoagulants that inhibit active coagulation factors in the bloodstream are distinguished by a specific inactivation pattern. Certain classes of substances, for example unfractionated, high molecular weight heparins, inhibit both thrombin and factor Xa. Other substances act highly specifically, thus inhibiting either thrombin (e.g. hirudin, dabigatran, melagatran) or factor Xa (e.g. pentasaccharides such as fondaparinux, rivaroxaban).
Direct and indirect inhibitors of the central procoagulatory factors of the blood coagulation system, factor Xa and thrombin, increasingly play a part in the treatment and prevention of cardiovascular and thromboembolic disorders. These inhibitors can be detected by the currently established assays only in a very complex manner. A simple and sensitive assay for said substances would be important both for therapy monitoring and for detecting the presence of said substances in an unknown patient sample. These assays should be able to detect a relatively high number of structurally unrelated thrombin or F Xa inhibitors in a highly sensitive manner.
The chromogenic assays currently used for determining anticoagulants involve mixing the patient sample to be analyzed, which usually consists of plasma, with a substrate for a coagulation factor. Since most blood coagulation factors are serine endopeptidases, i.e. hydrolases which can cleave peptide bonds, use is made mainly of peptide substrates which are cleaved as specifically as possible by the blood coagulation factor to be determined and which have a detectable signal group. The established chromogenic assays which are also available commercially employ in particular the chromophores para-nitroaniline (pNA) and 5-amino-2-nitrobenzoic acid (ANBA), which have an absorbance peak at 405 nm. The yellow color produced is normally determined photometrically. With the determination of anticoagulants, the color concentration in the assay mix is inversely proportional to the anticoagulant concentration in the sample.
In a first group of currently applied chromogenic assays for determining anticoagulants which inhibit the activity of blood coagulation factors, the patient sample to be tested is usually admixed with a defined amount of an activated coagulation factor and with a substrate for this coagulation factor. The more anticoagulant is present in the sample, the more inhibition of the activated coagulation factor takes place and the less substrate is cleaved. Examples of commercially available assays based on this assay principle are the Berichrom® heparin assay by Siemens Healthcare Diagnostics for determining heparin on the basis of inhibition of added factor Xa, or the hirudin activity assay by Siemens Healthcare Diagnostics for determining hirudin on the basis of inhibition of added thrombin.
In a second group of currently applied chromogenic assays for determining anticoagulants, the patient sample to be tested is admixed with an inactive coagulation factor, a coagulation activator and with a chromogenic substrate for the coagulation factor. Addition of the coagulation activator initially activates the inactive coagulation factor added. The more anticoagulant is present in the sample, the more inhibition of the activated coagulation factor takes place and the less substrate is cleaved. Examples of commercially available assays based on this assay principle are the Haemosys®-ECA T assay from JenAffin for determining synthetic direct thrombin inhibitors, or the Haemosys®-ECA H assay from JenAffin for determining hirudin on the basis of inhibition of thrombin/meizothrombin which is formed in the sample due to the addition of prothrombin and ecarin as coagulation activator.
EP-A1-2 177 625 describes another assay principle of determining anticoagulants. This assay principle likewise involves determining the cleavage of a coagulation factor-specific substrate, but with the aid of a different kind of signal-producing system rather than with the aid of chromogenic substrates. Said system comprises two components which, when binding simultaneously to the intact substrate, interact due to close spatial proximity and generate a detectable signal, for example fluorescence or chemiluminescence. As a result of the cleavage of a peptide bond of the substrate, the two components are separated from one another, and therefore no signal is produced. The more anticoagulant is present in the sample, the more inhibition of the activated coagulation factor takes place, the less substrate is cleaved and the more signal is produced. The advantage of fluorescence- or chemiluminescence-based assays over the chromogenic assays is basically that they are more sensitive and also allow measurements in whole blood.
The method described above in which a cleavable substrate is to a certain extent “mounted” between the two components of the signaling system is disadvantageous in that the substrate ligand on both sides of the cleavage site must have regions for association with the signaling components. The substrate ligands are usually synthetic, low molecular weight peptide substrates which have two artificial residues for association with the signaling components, for example an amino terminal Flag tag and a carboxy terminal biotin residue. However, coupling of such residues to low molecular weight peptide substrates always bears the risk of altering the structure of the peptide in such a way that it is no longer bound or not cleaved by the enzyme to be detected, and providing suitable substrate ligands is therefore technically complex.
It is thus desirable to modify known methods of determining anticoagulants, which use two components interacting due to close spatial proximity and generating a detectable signal, in such a way that low molecular weight ligands can be used which need to have no more than one artificial residue for association with any of the signaling components.
This object is achieved by admixing an aliquot of a sample which is suspected of containing an anticoagulant with a defined amount of a proteolytically active coagulation factor and with a ligand which binds to the active site of the proteolytically active coagulation factor but is not cleaved thereby at a peptide bond, and measuring the signal generated by the signal-producing system as a result of binding of the ligand to the activated coagulation factor. The inhibitor of the activated coagulation factor, i.e. the anticoagulant, present in the sample competes in a concentration-dependent manner with the ligand for binding to the active site of the proteolytically active coagulation factor and consequently inhibits signal generation. The more anticoagulant is present in the sample, the less signal is generated.